Abstract: Objective: To meet the requirements of laboratory quality control and proficiency testing in enteroaggregative Escherichia coli (EAEC) detection, homogenous and stable reference materials for enteroaggregative E. coli cells and genomic DNA (gDNA), with independent intellectual property rights, were developed. Methods: Next-generation sequencing (NGS) was used for whole genome sequencing of enteroaggregative E. coli (CMCC 44841). Bioinformatics analysis was performed on the sequencing results for species and virulence genes identification, serotyping, and multi-locus sequence typing (MLST). The characteristic genes of astA, aggR and pic were confirmed by PCR. Freeze-dried reference materials containing 103 CFU of cells and 20 ng of gDNA per sample were prepared. According to CNAS-GL017, homogeneity testing was performed on the 20 EAEC samples, and the results were statistically analyzed by one-way analysis of variance (ANOVA). The samples were stored at ?20, 4, 25 or 37 ℃ to evaluate their stability during storage and transportation. Then, three laboratories were organized for collaborative calibration and verification, and the applicability of the reference materials was tested on five different ready-to-eat food matrices according to the national standard method. Results: The genome size of CMCC44841 was 5.057 285 Mb, the GC content was 50.6%, and the genome contained 5 173 coding genes. The strain was identified as E. coli, serotyped as O127:H21, and typed as ST40 by MLST, carrying other virulence genes apart from aggR, astA and pic. The PCR fragments of astA, aggR and pic were 102, 400 and 1 111 bp in size, respectively. The result of homogeneity testing was F = 1.59, which meets the requirements for standard materials. The EAEC cell and gDNA samples were stable at ?20 and 4 ℃ for 60 days, at 25 ℃ for 7 days, and at 37 ℃ for 5 days. The collaborative interlaboratory calibration confirmed the EAEC cell reference material to contain 103 CFU of cells. The reference materials could be detected when they were added to 20 sample of different food matrices. Conclusion: The EAEC cell and gDNA reference materials, prepared from isolates obtained in the country, contain clear whole genome sequence information. They have good homogeneity, stability and applicability and can meet the requirements of quality control and proficiency testing in food testing laboratories.

Key words: diarrheagenic Escherichia coli; enteroaggregative E. coli; genomic DNA; reference material; whole genome sequencing; virulence gene